Globally, oral cancer is the sixth common malignancy with about 500,000 new oral and pharyngeal cancers diagnosed annually (Parkin, Pisani, & Ferlay, 1993, Estimates of the worldwide incidence of eighteen major cancers in 1985. Int J Cancer, 54(4), 594-606), and three quarters of these are from the developing world. At the Tata Memorial Hospital (Dinshaw & Ganesh, 2005 Annual Report—2001, Hospital Based Cancer Registry, Tata Memorial Hospital), Mumbai, which registers ˜30,000 cancer cases from all across the country, cancer of the oral cavity constitutes 12% of the total cancer load. Cancers of the buccal mucosa, which is a major site in the gingivo-buccal complex, are 59% of the oral cavity. Most of these cancers present at stage III and IV. The five-year survival is very low and about 60% patients return with loco-regional recurrence.
Molecular profiling of tumors is perceived as a tool for identifying prospective prognosticators or drug targets, with translational potential. For cancers of the head and neck, alterations in genes, which correlate with the now well-accepted hallmarks of cancer i.e. unregulated cell proliferation, reduced apoptosis, immortality, invasion and metastasis, and angiogenesis have been documented (Hunter, Parkinson, & Harrison, 2005 “Profiling early head and neck cancer. Nat Rev Cancer, 5(2), 127-135; Nagpal & Das, 2003 “Oral cancer: reviewing the present understanding of its molecular mechanism and exploring the future directions for its effective management”. Oral Oncol, 39(3), 213-221; Patel, Leethanakul, & Gutkind, 2001 “New approaches to the understanding of the molecular basis of oral cancer” Crit. Rev Oral Biol Med, 12(1), 55-63; Schliephake, 2003 “Prognostic relevance of molecular” markers of oral cancer—a review. Int J Oral Maxillofac Surg, 32(3), 233-245; Warnakulasuriya, 2002 “In Genetics of Human Cancer, Chapter 51, 773-784). Literature reports show inconsistency in their clinical relevance (Schliephake, 2003 “Prognostic relevance of molecular” markers of oral cancer—a review. Int J Oral Maxillofac Surg, 32(3), 233-245). A comprehensive analysis of oral cancer microarray data in literature by Shillitoe [www.upstate.edu/microb/shillite/Microarray_Oral_Cancer Genes.HTM] shows that the irreproducibility in the alterations in mRNA expression is at 93%. Studies in both the compilations have not taken into consideration subsites of the oral cavity, differences in methodology, sample size and extent of tumor cell representation in the specimens. Even with these limitations the array data is being pursued to provide information which can be utilized for cancer management (Sotiriou, Lothaire, Dequanter, Cardoso, & Awada, 2004” Molecular profiling of head and neck tumors. Curr Opin Oncol, 16(3), 211-214). From these earlier studies, it is becoming increasingly apparent that identification of site-specific molecular profiles is a must for diagnosis and/or prognosis.
Proteomic analysis using two dimensional gel electrophoresis-mass spectrometry (2DE-MS) has been reported for cancer of the buccal mucosa (Chen, He, Yuen, & Chiu, 2004 “Proteomics of buccal squamous cell carcinoma: the involvement of multiple pathways in tumorigenesis. Proteomics, 4(8), 2465-2475), oral squamous cell carcinoma (OSCC) (Lo et al., 2007“Identification of over-expressed proteins in oral squamous cell carcinoma (OSCC) patients by clinical proteomic analysis.” Clin Chim Acta, 376(1-2), 101-107.) and cancer of the tongue (Baker et al., 2005 “Proteome-wide analysis of head and neck squamous cell carcinomas using laser-capture microdissection and tandem mass spectrometry”. Oral Oncol, 41(2), 183-199; He, Chen, Kung, Yuen, & Chiu, 2004 “Identification of tumor-associated proteins in oral tongue squamous cell carcinoma by proteomics”. Proteomics, 4(1), 271-278). In the study with cancers of buccal mucosa (Chen et al., 2004 “Proteomics of buccal squamous cell carcinoma: the involvement of multiple pathways in tumorigenesis”. Proteomics, 4(8), 2465-2475), the differences in protein expression between normal and tumor tissue was investigated using whole tissue samples; the tumor tissue comprising of about 70% tumor cells and normal tissue with <15% of epithelium and the rest with muscle and surrounding stroma. In the study with OSCC (Lo et al., 2007 “Identification of over-expressed proteins in oral squamous cell carcinoma (OSCC) patients by clinical proteomic analysis”. Clin Chim Acta, 376(1-2), 101-107), the tumor tissue used contained >90% tumor cells. The percentage of normal epithelium was not defined. Baker et al (Baker et al., 2005 “Proteome-wide analysis of head and neck squamous cell carcinomas using laser-capture microdissection and tandem mass spectrometry”. Oral Oncol, 41(2), 183-199.) have reported a protein profile for squamous cell carcinoma (SCC) of the tongue obtained after laser capture microdissection followed by LC-MS/MS analysis. The relative abundance of a protein in normal and tumor epithelium was quantified by the number of times a protein was identified in each of them. Gires et at (Gires et al., 2004 “Profile identification of disease-associated humoral antigens using AMIDA, a novel proteomics-based technology”. Cell Mol Life Sci, 61(10), 1198-1207.) and Rauch et al (Rauch et al., 2004 “Allogenic antibody-mediated identification of head and neck cancer antigens”, Biochem Biophys Res Commun, 323(1), 156-162.) have identified oral cancer-specific antigens eliciting immune response in patients. However, in these studies, much of the work is with tumor cell lines and head and neck tumors with no subsites defined. In none of the above studies, the relevance of the co-expression and the ability of a set of differentially expressed proteins, to distinguish between normal, benign, and transformed epithelium has been evaluated. In a very recent study (Roesch-Ely et al., 2007 “Proteomic analysis reveals successive aberrations in protein expression from healthy mucosa to invasive head and neck cancer”. Oncogene, 26(1), 54-64.) proteomic analysis using SELDI-TOF-MS has revealed successive aberrations in protein expression from healthy mucosa to invasive head and neck cancer. In this study, however, whole tissue with tumor cells ranging from 40%-90% from different head and neck sites were used.
Our study analyses proteomic profiles of squamous cell carcinoma (SCC) of the gingivo buccal complex (GBC) and adjacent clinically non-malignant tissue from the same individuals using manually dissected epithelium. A set of differentiator proteins has been generated from relative quantitative assessment of two dimensional gel electrophoresis profiles of the dissected normal and tumor epithelia. This is the first time, for cancer of an oral subsite that co-expression of eleven proteins was found to distinguish the transformed epithelium from the normal, with good specificity and sensitivity, as assessed by cluster analysis and further confirmed with Receiver Operator Characteristics (ROC) analysis. The minimal numbers of proteins which are able to differentiate non malignant and malignant epithelial tissue were further assessed by Linear Discriminant analysis and the identity of the differentiator proteins was obtained by mass spectrometry and validated by western blotting of the total protein lysates from microdissected tissues.
Out of the proteins identified, gamma actin, HSP27, triosephosphate isomerase, GST π, 14-3-3 σ and tropomyosin were reported earlier (Chen et al., 2004 “Proteomics of buccal squamous cell carcinoma: the involvement of multiple pathways in tumorigenesis. Proteomics, 4(8), 2465-2475; Lo et al., 2007 “Identification of over-expressed proteins in oral squamous cell carcinoma (OSCC) patients by clinical proteomic analysis”. Clin Chim Acta, 376(1-2), 101-107; Roesch-Ely et al., 2007 “Proteomic analysis reveals successive aberrations in protein expression from healthy mucosa to invasive head and neck cancer”. Oncogene, 26(1), 54-64.) in oral epithelium, while lactate dehydrogenase, prohibitin, cathepsin D, thioredoxin peroxidase, apolipoprotein A-I, tumor protein translationally controlled-1, an SFN family protein and 14-3-3 ζ (YWHAZ), are being reported for the first time in the epithelium of the gingivo buccal complex by proteomic studies. The differentiators among these are lactate dehydrogenase, alpha enolase, prohibitin, cathepsin D, apolipoprotein A-I, tumor protein translationally controlled-1, an SFN family protein, 14-3-3σ tropomyosin, protein spot 81 {14-3-3ζ(YWHAZ)} and protein spot 57a for which identity is still to be obtained. Linear Discriminant analysis has additionally revealed that 14-3-3σ, lactate dehydrogenase and apolipoprotein A-I are key discriminants of the transformed epithelium and could serve as potential markers or targets for therapy.